WO1998052034A1 - Pompe a expansion thermique pour ecoulement de liquide sans impulsion - Google Patents

Pompe a expansion thermique pour ecoulement de liquide sans impulsion Download PDF

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Publication number
WO1998052034A1
WO1998052034A1 PCT/US1998/010119 US9810119W WO9852034A1 WO 1998052034 A1 WO1998052034 A1 WO 1998052034A1 US 9810119 W US9810119 W US 9810119W WO 9852034 A1 WO9852034 A1 WO 9852034A1
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WO
WIPO (PCT)
Prior art keywords
liquid
tubing
temperature
capillary
thermal expansion
Prior art date
Application number
PCT/US1998/010119
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English (en)
Inventor
Stellan Hjerten
Christer Ericson
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Bio-Rad Laboratories, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bio-Rad Laboratories, Inc. filed Critical Bio-Rad Laboratories, Inc.
Priority to AU75771/98A priority Critical patent/AU7577198A/en
Publication of WO1998052034A1 publication Critical patent/WO1998052034A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F04—POSITIVE - DISPLACEMENT MACHINES FOR LIQUIDS; PUMPS FOR LIQUIDS OR ELASTIC FLUIDS
    • F04B—POSITIVE-DISPLACEMENT MACHINES FOR LIQUIDS; PUMPS
    • F04B19/00—Machines or pumps having pertinent characteristics not provided for in, or of interest apart from, groups F04B1/00 - F04B17/00
    • F04B19/20—Other positive-displacement pumps
    • F04B19/24—Pumping by heat expansion of pumped fluid
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F04—POSITIVE - DISPLACEMENT MACHINES FOR LIQUIDS; PUMPS FOR LIQUIDS OR ELASTIC FLUIDS
    • F04F—PUMPING OF FLUID BY DIRECT CONTACT OF ANOTHER FLUID OR BY USING INERTIA OF FLUID TO BE PUMPED; SIPHONS
    • F04F1/00—Pumps using positively or negatively pressurised fluid medium acting directly on the liquid to be pumped
    • F04F1/02—Pumps using positively or negatively pressurised fluid medium acting directly on the liquid to be pumped using both positively and negatively pressurised fluid medium, e.g. alternating
    • F04F1/04—Pumps using positively or negatively pressurised fluid medium acting directly on the liquid to be pumped using both positively and negatively pressurised fluid medium, e.g. alternating generated by vaporising and condensing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • G01N2030/326—Control of physical parameters of the fluid carrier of pressure or speed pumps

Definitions

  • This invention lies in the field of liquid flows, including both micro volume flows such as those used in capillary chromatography, and larger volume flows, and addresses in particular the difficulties encountered in attempting to achieve a steady, pulse-free flow of liquid at very low flow rates.
  • capillaries for separations and analyses of multi-component mixtures
  • capillaries offer the ability to analyze very small sample volumes, to perform analyses in a relatively short period of time but with high reproducibility and accuracy, and to perform on-line detection, and the use of cartridges to hold the capillaries and to permit easy exchange of one separation medium or column for another, the cartridges lending themselves readily to use in automated systems for the analysis of a multitude of samples in succession.
  • the means by which the mixture components are separated can vary widely, reflecting the many different types and sources of mixtures that require analysis, the variety of separation media needed to separate the components, and the variety of chromatographic methods that have been developed to achieve these separations.
  • Most powerful group of separation techniques are those involving high performance liquid chromatography (HPLC). Included in this group are ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, molecular sieve chromatography, adsorption chromatography, and exclusion chromatography. In these techniques, the mobile phase is forced through the separation medium by the mechanical force of a pump.
  • the performance of the pump is a particularly significant factor in the separation.
  • the flow pulses created by the mechanical action of the pump disrupt the detector baseline to an increasing extent, interfering with the separation results.
  • One means of reducing this interference is by splitting the pump discharge so that only a portion of the flow enters the capillary. With this method, the pump can be run at a higher flow rate than that directed through the capillary, thereby reducing the pulsing.
  • the disadvantages are that the flow rate through the column is not known with certainty, and the excess carrier fluid that by-passes the column is wasted.
  • a thermal expansion pump which includes a non-expandable (rigid-walled) vessel that is filled with a liquid that undergoes volumetric expansion upon heating, plus a heating device for heating the vessel and its contents uniformly and at a highly controlled rate.
  • the vessel is completely filled with the liquid and fully closed except for one outlet port for discharge of the liquid, such that all volumetric expansion of the liquid is discharged through the outlet port upon heating.
  • the vessel can be a bomb, a tank, a length of tubing, or any container that is capable of retaining and being completely filled with liquid and does not expand upon increases in the pressure of the liquid.
  • a preferred heating device is a temperature bath under computer control monitored by a temperature sensor in the bath.
  • the tubing When the vessel is a length of tubing, the tubing is closed at one end and open at the other, and the temperature rise creates a discharge of the expanding liquid from the open end of the tubing at a controlled rate. Rapid equalization of temperature inside the tubing is achieved by using tubing of relatively small diameter and thin-wall construction. When the vessel is a tank or bomb, rapid equalization of temperature of the liquid is achievable by stirring, or other means of agitation of the vessel or its contents. In general, substantial equalization of temperature should be obtained within about 5 seconds, and preferably within about one second.
  • the preferred vessel is a length of tubing.
  • the discharge end of the tubing is connected to the capillary column, either directly or through an intermediate length of tubing or any enclosed reservoir that will transmit the expansion flow from the temperature-rise tubing to the capillary.
  • sample injection will be performed at the entry end of the capillary.
  • the temperature-rise tubing will contain the carrier liquid (generally a buffer solution or when desired a gradient buffer solution) that will draw the sample through the capillary.
  • the carrier liquid can be initially retained in the intervening tubing or reservoir while the liquid in the temperature- rise tubing can be any liquid that expands volumetrically upon heating.
  • Intervening tubing is particularly useful in applications where a concentration gradient, such as a salt gradient, for example, is desired.
  • the invention is useful in achieving a wide range of flow rates, but will be of particular interest in capillary HPLC where the flow rate produced is 10 ⁇ L/min (microliters per minute) or less.
  • the invention will also be of interest in capillaries containing a continuous solid yet porous polymeric bed as a separation medium.
  • FIG. 1 is a plot of the coefficient of volumetric thermal expansion vs. temperature for water.
  • FIG. 2 is a diagram of a thermal expansion pump and capillary chromatography column utilizing the principles of the present invention.
  • FIG. 3 is a plot of flow rate vs. temperature rise/minute for a thermal expansion pump of the present invention.
  • FIG. 4 is a plot of flow rate vs. pressure for a thermal expansion pump of the present invention.
  • FIG. 5 is a chromatogram of a protein mixture taken on a cation exchange column using a thermal expansion pump of the present invention as the carrier liquid driving force.
  • FIG. 6 is a chromatogram of a protein mixture taken on a smaller diameter cation exchange column, again using a thermal expansion pump of the present invention as the carrier liquid driving force.
  • FIG. 7 is a chromatogram of a protein mixture taken on a hydrophobic interaction column using a thermal expansion pump of the present invention as the carrier liquid driving force.
  • FIGS. 8a and 8b are records of pressure vs. time at two different pressures and flow rates for a conventional HPLC piston pump.
  • FIGS. 9a and 9b are records of pressure vs. time at four different flow rates for a thermal expansion pump of the present invention.
  • FIG. 10 is a diagram of a thermal expansion pump and tubular chromatography column utilizing the principles of the present invention to achieve a higher rate of flow than that of the apparatus of FIG. 2.
  • This invention is primarily of interest in achieving volumetric flow rates with the range of about 0.001 ⁇ L per minute to about 100 ⁇ L per minute, although larger flow rates can also be achieved, depending on the nature of the temperature rise vessel.
  • Preferred flow rates for certain applications, notably HPLC are those in the range of about 0.3 ⁇ L/min to about 10 ⁇ L/min, and for extremely small capillaries, those in the range of about 0.001 ⁇ L/min to about 0.03 ⁇ L/min.
  • Selection of the flow rate is achieved by a combination of factors, including the volumetric capacity (length and internal diameter) of the temperature-rise tube, the rate of temperature rise, and the coefficient of thermal expansion of the driving liquid retained in the temperature-rise tube.
  • the volumetric coefficient of thermal expansion ⁇ for a liquid at a given temperature T is defined as
  • V is the volume of the liquid and Tlbt is the rate rise of the temperature of the liquid in degrees Celsius per second. Since V is constant and ⁇ for typical liquids varies in a monotonic way with temperature, the result of imposing a controlled temperature rise is a pulse-free flow.
  • any liquid that expands volumetrically with an increase in temperature and for which a substantially constant rate of expansion can be achieved by a continuous temperature increase can be used.
  • Coefficients of thermal expansion and their values as a function of temperature are known and are generally available in the literature. Although the particular value of the coefficient is not critical to this invention, best results in most cases will be obtained with a liquid having a coefficient of about 1 X 10 "4 K "1 or greater at 1 atmosphere and 20°C, preferably within the range of about 1 x 10 ⁇ 4 K "1 to about 20 X 10" 4 K” 1 at 1 atmosphere within a temperature range of about 20°C to about 100°C. Water is one example of a liquid useful for this purpose, and its coefficient of thermal expansion as a function of temperature is shown graphically in FIG. 1.
  • the compressibility of the driving liquid is a further factor in the performance of the thermal expansion pump of this invention.
  • the compressibility of a liquid at the temperature T and pressure P can be expressed in terms of the compressibility factor ⁇ P , which is defined as
  • the vessel can be a tank, bomb or other non- tubular vessel, with a volumetric capacity of 100 mL or greater, preferably from about 100 mL to about 10 L.
  • a stirrer such as a magnetic stirrer can be used to achieve rapid equalization of the temperature.
  • the vessel can be a length of tubing, with inner diameter of abot 1.0 cm or less and volumetric capacity of 100 mL or less, and preferably and inner diameter within the range of about 0.01 mm to about 1.0 cm and a volumetric capacity within the range of about 0.01 mL to about 100 mL.
  • the driving liquid can be the same liquid entering the capillary where chromatographic separation is being conducted, but it is preferred that an intermediate reservoir or length of tubing separate the temperature rise vessel from the capillary, such that the driving liquid does not enter the capillary. In this way, the capillary is isolated from the temperature rise in the driving liquid.
  • the driving liquid can be one that is either miscible or partially or totally immiscible with the carrier liquid passing into the capillary.
  • the intermediate reservoir or length of tubing must be configured or arranged such that only the carrier liquid enters the capillary.
  • the temperature rise vessel can be of any material that will retain the thermal expansoin liquid and that will not expand to any substantial degree in response to an increase in pressure of the liquid retained inside that is likely to be encountered during the operation of the pump (t ' .e. , excluding increases in pressure caused by vaporization).
  • a temperature rise vessel that is a length of tubing
  • the temperature rise tube when in operation is closed at one end so that volumetric expansion will drive the expanding liquid from the other end only.
  • Metals such as stainless steel as well as other materials such as silica capillaries can be used for the temperature rise tube.
  • the inner diameter of the tubing can vary, and will be selected according to the needs of the system, as indicated above.
  • the volumetric capacity of the tubing can likewise vary, depending on the needs of the system as indicated above. Preferred ranges are given above, and further preferred ranges are a volumetric capacity of from about 0.03 mL to about 30 mL and an inner diameter of from about 0J mm to about 3.0 mm.
  • the wall thickness of the tubing is also a factor, and will be selected accordingly. In most applications, the wall thickness will range from about 10 microns to about 1,000 microns.
  • the rate of temperature rise can vary as well, and will be selected according to the desired flow rate. In most cases, the temperature rise vessel and the driving liquid will be selected such that an appropriate rate of temperature rise will be from about 0.05 to about 20 degrees Celsius per minute, and preferably from about 0.1 to about 5 degrees Celsius per minute.
  • the temperature rise is achieved by any conventional means that is susceptible to a high level of control and rates within the ranges cited above.
  • immersion of the tubing in a thermally controlled liquid bath is a preferred method, particularly a temperature bath whose temperature is continually monitored and adjusted by computer.
  • the temperature can be controlled by a heating coil in the tank or bomb interior, either electrically heated or having a heat-transfer fluid passing through the interior of the coil. In general, however, any heating device that will provide controlled heating at the desired rate can be used.
  • thermal expansion pump of this invention is of use in any application where a pulse-free flow of liquid at a highly controlled yet very slow rate is needed, the invention is of particular interest in capillary chromatography.
  • the pump is useful with any kind of chromatographic bed, including those in which the bed is a continuous solid porous bed that has been formed in the capillary itself.
  • An example of one such bed is that formed from a polymerization reaction mixture containing one or more water-soluble polymerizable compounds such as vinyl, allyl, acrylic and methacrylic compounds, and a crosslinking agent, and ammonium sulfate, in amounts such that:
  • the amount concentration of ammonium sulfate in the polymerization reaction mixture is within the range of about 1.8 to about 4.0 M.
  • polymerizable compounds are vinyl acetate, vinyl propylamine, acrylic acid, butyl aery late, acrylamide, methacrylamide, glycidyl methacrylate, glycidyl acrylate, methylene-bis-acrylamide, and piperazine diacrylamide.
  • ion exchange columns charged compounds, due to the inclusion of functional groups, will be included.
  • Examples of functional groups for anion exchangers are quaternary ammonium groups with either three or four alkyl substitutions on the nitrogen atom, the alkyl groups being primarily methyl or ethyl, and in some cases themselves substituted, for example with hydroxyl groups.
  • Examples of functional groups for cation exchangers are sulfonic acid groups and carboxylic acid groups, joined either directly to the resin or through linkages.
  • Examples of functional groups for reversed phase chromatography are octylacrylate and octadecylacrylate.
  • the crosslinking agent(s) will be selected in accordance with the monomers.
  • suitable crosslinking agents are bisacrylamides, diacrylates, and a wide range of terminal dienes. Specific examples are dihydroxyethylenebisacrylamide, diallyltartardiamide, triallyl citric triamide, ethylene diacrylate, bisacrylylcystamine, N,N'-methylenebis- acrylamide and piperazine diacrylamide. Appropriately derivatized, the column can be used for anion exchange, cation exchange, hydrophobic interaction, affinity, or reversed phase chromatography.
  • a thermal expansion pump in accordance with this invention was constructed as shown in FIG. 2, where it is shown with a capillary column whose flow rate is controlled by the pump.
  • Components of the pump 11 are a water bath 12 with built-in heater and thermostat, a computer 13 regulating the temperature of the water bath based on the thermostat potential, a spiral stainless steel tube 14 24.9 m in length, with inner diameter 0.75 mm and outer diameter 1.59 mm and an internal volume of 11.00 mL, and a pressure gauge 15.
  • the stainless steel tube was replaced with a coiled fused silica capillary of length 1.37 m, inner diameter 320 microns, and outer diameter 405 microns.
  • a control valve 16 consisting of a three-port high-pressure HPLC valve was connected to the pump discharge line, the valve containing two open-and-close ports 17, 18 and a titanium rotor 19 to increase or decrease the internal volume of the control valve.
  • a Teflon tube 20 used in the formation of salt gradients joined the control valve output to a three-port switching valve 21.
  • the switching valve led to a capillary column 22, a UV detector 23, and a precision micro-scale balance 24. Also shown in the drawing is a syringe 25 for drawing fluids through the system, as explained below.
  • the inner diameter of the gradient tubing 20 was selected in accordance with the inner diameter of the capillary column 22: for a 320 ⁇ m capillary column, gradient tubing with an inner diameter of 250 ⁇ m was used; for a 15 ⁇ m capillary column, gradient tubing with an inner diameter of 25 ⁇ m was used.
  • the micro-scale balance was Model AE 260-S from Mettler-Toledo AG (Gsammlungsee, Switzerland), and was used for obtaining measurements of the flow rate through the capillary column 22.
  • the balance weighed the column eluent collected in a glass vial 26, that was fed by a fused silica capillary (25 micron inner diameter) leading from the outlet end of the capillary column and passing into the vial through a hole in the cap of the vial.
  • the hole was 0.5 mm in diameter to prevent evaporation of the column eluent.
  • a bubble flow meter was constructed by mounting a 10-cm long transparent glass tube (1.0 mm inner diameter) to a horizontal ruler and joining the tube to the capillary outlet by a press-fit connector. Both the glass tube and the connector were coated with vinyltrichlorosilane to lower the affinity of the tube and connector to water. Flow rates were determined by measuring the time required for the meniscus to travel between two tbin lines on the tube. Determination of Temperature Rise/Flow Rate Relation
  • a stable pressure and flow rate were achieved within 10-20 seconds.
  • the need for this equilibration time can be attributed to elasticity in the system, including the gradient tubing 20, a lag time in the interface between the computer 13 and the water bath 12, or both. While this affords the advantage of eliminating abrupt increases in pressure, a faster rate of increase of the pressure and flow rate can be obtained by adjusting the rotor 19 to lower the internal volume in the control valve 16.
  • Equation (2) The rate of temperature increase ⁇ 77 ⁇ t required to deliver a particular flow rate AV/ ⁇ t using the stainless steel tube was calculated by Equation (2) above, and compared to actual flow rates measured by the micro-scale balance with an eluent temperature of 25°C (density 0.997 g/cm 3 ). A temperature rise of from 41°C to 52°C was performed at rates ranging from 0.28°C/min to 2.20°C/min, in each case displacing a total volume of 52.4 ⁇ L.
  • the value of ⁇ ⁇ used in Equation (2) was 4.237 x 10 "4 KJ, which is the mean of ⁇ values from the literature (Handbook of Chemistry and Physics, 68th ed., Robert C.
  • the thermal expansion pump (with the stainless steel coil) was used to deliver a 20 mM sodium phosphate buffer, pH 6.2, to the columns over a range of flow rates, and back pressures were measured for each flow rate.
  • the results are plotted in FIG. 4, where the "A" curve, with diamond-shaped points, represents the lower back-pressure column, and the "B" curve, with circular points, represents the higher back-pressure column.
  • the plot shows that both columns gave a linear relation between the flow rate and the back pressure.
  • the larger scattering of points at lower flow rates reflects the relatively small differences in the masses of the effluent fractions and the difficulties in making accurate measurements of these differences.
  • Continuous bed separation media were prepared in a fused silica capillary column 140 mm in length (110 mm effective length), with inner diameter 320 ⁇ m and outer diameter 405 ⁇ m with an external polyimide coating, by the following procedures.
  • a short section of the polyimide coating was burned off of the capillary surface to serve as a window for on-line detection.
  • the capillary interior was then washed with toluene and then acetone, then treated for 30 minutes with 0.2 M NaOH, followed by 30 minutes with 0.2 M HCl, and finally rinsed with distilled water.
  • the capillary wall was then activated with 3-methacryloyloxypropyl trimethoxysilane in acetone (30% by volume) to place methacryloyl acid residues on the internal capillary wall.
  • Each monomer solution was degassed with a stream of nitrogen and supplemented with 10 ⁇ L of 10% ammonium persulfate and 10 ⁇ L of 5% aqueous TEMED (N,N,N,N- tetramethylethylenediamine) -.oiution befor : being aspirated into the activated column. Polymerization proceeded for 24 hours.
  • This example illustrates the use of the thermal expansion pump of the present invention as the driving force for cation exchange chromatography, using a linear salt gradient.
  • a 48- ⁇ L linear salt gradient extending from 0 to 0.6 M NaCl in 20 mM sodium phosphate, pH 6.2 was prepared as follows. Sixteen (16) solutions were prepared in test tubes by mixing solution A (20 mM sodium phosphate, pH 6.2) with solution B (0.6 M NaCl in 20 mM Na ⁇ PO,,, pH 6.2) in different proportions, the volume percentage of solution B increasing in equal increments from 0% in the first test tube to 100% in the sixteenth test tube. Using the system shown in FIG. 1, the capillary column 22 was equilibrated with solution A delivered by the thermal expansion pump 11, with the switching valve 21 in position to direct the flow from the pump into the column.
  • the switching valve was then turned to permit fluid to be drawn backward into the gradient tubing 20 through the input line 31, and by means of the syringe 25, 3- ⁇ L portions from each of the sixteen test tubes in succession, beginning with the sixteenth test tube (100% solution B) and ending with the first (100% solution A) were then drawn into the gradient tubing 20 to form the 48- ⁇ L positive salt gradient in the tubing.
  • the syringe 25 was used to draw a 1- ⁇ L aliquot of a protein test mixture into the tubing 20 (through the input line 31, with the switching valve 21 connecting the input line 31 to the gradient tubing 20), followed by 4 ⁇ L of the starting buffer (i.e., an additional 4 ⁇ L of the last of the sixteen buffers to be drawn into the tubing).
  • the switching valve 21 was then redirected toward the capillary column 22, and the syringe 25 was replaced with a finger-tight plug.
  • the chromatography experiment was then begun by starting the selected temperature rise in the water bath 12 and opening the control valve 16 by slowly turning the rotor 19 upwards.
  • the protein mixture consisted of 0.4 mg/mL each of myoglobin (horse), cytochrome C, and lysozyme, plus 1.2 mg/mL of ribonuclease.
  • the coil in the thermal pump was the stainless steel coil described above, and the pump was operated with a programmed linear temperature gradient of 1.25°C/min, starting at 41 °C, and a constant pressure of 30 bar (measured), producing a flow rate of 5.8 ⁇ L/min.
  • the capillary column was as described above, with 1 10 mm effective length and 320 ⁇ m inner diameter. Detection was performed at 280 nm wavelength. The resulting chromatogram is shown in FIG. 5, whei peak 1 represents myoglobin, peak 2 represents ribonuclease, peak 3 represents cytochrome C, and peak 4 represents lysozyme.
  • a second cation exchange chromatography run was performed on the same protein mixture, but with a smaller capillary column and a smaller coil in the thermal expansion pump.
  • the column was a fused silica capillary 140 mm in length (1 10 mm effective length), with inner diameter 15 ⁇ m and outer diameter 140 ⁇ m, treated and packed with a continuous bed in the same manner as the larger capillary.
  • the pressure coil in the thermal expansion pump was the coiled fused silica capillary 1.37 m in length with an internal diameter of 320 ⁇ m. This coil produced a volume change that was 100-fold less than that of the stainless steel tube.
  • the pump was operated with a programmed linear temperature gradient of 0.25°C/min, starting at 41°C, and a constant pressure of 28 bar (measured), producing a flow rate of 10 nL/min without splitting of the mobile phase.
  • the sample volume was 0.01 ⁇ L.
  • the resulting chromatogram is shown in FIG. 6.
  • the similarity in appearance between the chromatograms of FIGS. 5 and 6 is significant. Both show a clean separation of the individual proteins and high resolution for each peak.
  • This example illustrates the use of the thermal expansion pump of the present invention as the driving force for hydrophobic interaction chromatography, again using a linear salt gradient although a decreasing gradient rather than an increasing gradient.
  • the gradient mobile phase extended from 2.4 to 0 M (NH 4 ) 2 SO 4 in 20 mM
  • Na j PO,,, pH 6.8, was prepared in a manner analogous to that of the gradient mobile phase of Example 1, using the 2.4 M (NH 4 ) 2 SO 4 solution in one test tube, the solution lacking (NH 4 ) 2 SO 4 in another, and fourteen graduated mixtures of the two in intermediate test tubes, and loading small aliquots from each tube in succession into the gradient tubing 20.
  • the separation was then performed on a 1- ⁇ L protein mixture consisting of 0.07 mg/mL cytochrome C and 0.4 ⁇ L each of myoglobin (horse), ribonuclease, lysozyme, and ⁇ -chymotrypsinogen, using the hydrophobic interaction separation medium described above.
  • the capillary column had an effective length of 100 mm and an inner diameter of 320 ⁇ m.
  • the coil in the thermal pump was the stainless steel coil described above, and the pump was operated with a programmed linear temperature gradient of
  • peak 1 represents cytochrome C
  • peak 2 represents myoglobin
  • peak 3 represents ribonuclease
  • peak 4 represents lysozyme
  • peak 5 represents ⁇ -chymotrypsinogen. Complete separation and high resolution were achieved.
  • This example compares the pressure vs. time performance of the thermal expansion pump of the present invention with a convention HPLC piston pump.
  • the HPLC piston pump used in this comparison was Model No. 2150 obtained from LKB, Sweden.
  • a splitting capillary was utilized (i.e., the pump discharge was divided between two capillaries arranged in parallel). Since this arrangement permitted the pump to operate at a flow rate higher than that delivered to the individual capillaries, the splitting capillary provided the system with a further advantage by rendering the disturbances caused by the reciprocating motion of the pistons less pronounced than they would otherwise have been.
  • the thermal expansion pump the stainless steel coil described above was used.
  • FIGS. 8a and 8b The pressure traces are shown in FIGS. 8a and 8b for the HPLC piston pump and FIGS. 9a and 9b for the thermal expansion pump. These traces indicate that the pressure traces from the thermal expansion pump were almost free from noise and fluctuations at all flow rates and back pressures, and considerably more stable than those from the HPLC piston pump. Even at 50 ⁇ L/min (50 bar), which is a high flow rate for capillary chromatography, the pressure pulses were negligible. The steady pressure observed at flow rates of 1 ⁇ L/min and 10 nL/min is a prerequisite for efficient separations on very narrow bore columns.
  • FIG. 10 illustrates a variation on the apparatus of FIG. 2, designed to produce a higher- volume flow while still using the principles of the present invention.
  • the liquid 40 whose expansion causes the flow in the thermal expansion pump in this example is retained in an insulated tank 41 that is closed except for one discharge port 42. With the tank completely filled with liquid, all thermal expansion of the liquid in the tank causes flow through the discharge port.
  • the temperature of the liquid in the tank, and the rate of temperature rise, is controlled by a heating coil 43, in combination with a magnetic stirrer bar 44. Circulating inside the heating coil 43 is a heat transfer liquid, whose temperature is controlled by an external temperature control bath 45, similar to the temperature control bath 12 of FIG. 2.
  • the temperature of the temperature control bath 45 is controlled by a built-in thermostat and a computer 46 similar to the computer 13 of FIG. 2.
  • a circulation pump 47 causes the heat transfer liquid to circulate between the coil 43 in the thermal expansion liquid tank 42 and the coil 48 in the temperature control bath 45.
  • the volume of thermal expansion liquid 41 in the tank 41 is considerably greater than the volume of the thermal expansion liquid in the coil 14 of FIG. 2.
  • the same increase in temperature therefore produces a greater increase in volume although the proportional increase relative to the total volume of thermal expansion liquid in both cases will the same, assuming equal coefficients of thermal expansion.
  • the higher volumetric flow rate in FIG. 10 can be used for any application where a pulse-free flow is required; one example is the higher diameter chromatographic separation column 49 shown in the Figure. Detection of separated components is achieved by a conventional detector 50.

Abstract

La présente invention concerne un écoulement sans impulsion, par exemple pour chromatographie capillaire, produit par une pompe constituée d'une longueur de tube transmettant la chaleur, remplie d'un liquide présentant une expansion thermique sous l'effet de la chaleur et d'un dispositif de chauffage qui chauffe de manière uniforme le tube et son contenu de façon régulée.
PCT/US1998/010119 1997-05-15 1998-05-14 Pompe a expansion thermique pour ecoulement de liquide sans impulsion WO1998052034A1 (fr)

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AU75771/98A AU7577198A (en) 1997-05-15 1998-05-14 Thermal expansion pump for pulse-free liquid flow

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US85702497A 1997-05-15 1997-05-15
US08/857,024 1997-05-15

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Cited By (1)

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